The HLA complex is the most polymorphic genetic system known in man. The frequency of the HLA class II antigens have been well studied in Caucasoids but little data is available concerning HLA antigen frequencies in Negroes. In this thesis the class II antigens, excluding HLA-DP, were studied in South African (SA) Negroes (Xhosa), Cape Coloureds ( a group of mixed racial origin) and SA Caucasoids using serological, cellular ( HTC typing) and Southern blot techniques. The results obtained for the SA Negroes were compared with those previously found in Nigerians and American Negroes. Marked differences in HLA distribution occurred between these groups, which in part may be explained by Khoisan admixture in the SA Negroes. In addition, striking frequency differences were observed between the three SA populations. For example, in the Xhosa the HLA-DR1, DR4, DR7, DRw8, DQw2, DQw3, Dw1 and Dw3 specificities were found at a significantly lower frequency, whereas HLA-DR3, DRw6 and Dw' RSH' were found at a significantly higher frequency compared with the SA Caucasoids. The frequency in the Cape Coloureds was intermediate between those of the Xhosa and Caucasoids. In the SA Negroes and Cape Coloureds, several new specificities were detected such as HLA-DRw18, DR2 LUM(CT), DRwl2x6, DRw8x14, Dw' RSH', Dw' JOH' and Dw' BME'. The HLA-DR and DQ haplotypes in significant linkage disequilibrium were similar in the three groups. However, several haplotypes with unusual DR and DQ combinations such as HLA-DRw17,DQw7; DR9, DQw2 and DR4, DQw5 were present in the SA Negroes and Cape Coloured families. Al though some of these unusual haplotypes could be explained in terms of recombination between the common haplotypes, none could be typed using a panel of well defined homozygous typing cells, suggesting that the response observed in mixed lymphocyte culture arises from separate molecular determinants. The data on HLA class II antigen frequencies presented in this thesis is essential for future studies on HLA and disease associations and for establishing population relationships. Knowledge of new HLA class II antigens in the various population groups is also important in renal transplantation as matching for HLA-DR antigens is known to improve graft survival.

and Key words This diploma thesis is focused on assessing the potential benefit of HLA epitopes for the prediction of de novo antibody production at kidney transplant recipients. The topic and patient selection criteria were selected in accordance with the 18th International HLA and Immunogenetics workshop (IHIWS), which is taking place in May 2022 in the Netherlands, where our data will also be contributed. Our aims were to compare HLA antigens mismatches (counted as total number of mismatched alleles) defined on the high-resolution level by NGS sequencing, HLA eplets mismatches defined by HLA matchmaker, and amino acid mismatches defined by HLA EMMA in their capacity to predict de novo antibody production and compare these results to other works by different authors from this field. We have identified N= 28 patients who developed de novo antibodies and N= 19 who didn't develop de novo antibodies in 5 years follow up their transplant. These two cohorts were compared based on all three approaches and correlation between number of mismatches and number of patients with de novo antibodies were made using ROC curves. Superiority of eplet mismatches over HLA antigen mismatches (total number of mismatched alleles) defined on high resolution was not detected. The HLA epitopes identified by the HLA...

Bien que la greffe de cornée est le type de transplantation connaissant le plus haut taux de succès, le rejet est la principale cause d'échec de greffe, surtout chez les cornée vascularisées. Comme l'aspect immunologique est primordial pour les greffes d'autres organes, nous analysons le volet sérologique, avec l'effet du crossmatch, et le volet tissulaire, avec l'effet des antigènes ABO, Lewis, HLA -A, -B et DR, chez une population de patients greffés au CHUL entre 1982 et 1995. Le suivi minimal est de 12 mois. Le crossmatch positif chez des patients présensibilisés par une greffe antérieure ou un rejet antérieur augmente le risque de rejet endothelial. Encore une fois, nous démontrons que ABO n'influence pas la survie de la greffe. Une incompatibilité Lewis ou HLA -A ou -B augmente le rejet chez les patients non-vascularisés.L'effet d'un mismatch HLA-DR n'a pas été démontré. / Québec Université Laval, Bibliothèque 2018

Monocytic human leukocyte antigen-D related (mHLA-DR) expression determined by flow cytometry has been suggested as a biomarker of sepsisinduced immunosuppression. In order to facilitate use of HLA-DR in clinical practice, a quantitative real-time PCR technique measuring HLA-DR at the transcription level was developed and evalutated. Levels of HLA-DR mRNA correlated to mHLADR expression and were robustly measured, with high reproducibility, during the course of infection. Dynamics of mHLA-DR expression was studied during the first weeks of bloodstream infection (BSI) and was found to be dependent on the bacterial etiology of BSI. Moreover, mHLA-DR was shown to be inversely related to markers of inflammation. In patients with unfavourable outcome, sustained high C-reactive protein level and high neutrophil count were demonstrated along with low mHLA-DR expression and low lymphocyte count. This supports the theory of sustained inflammation in sepsis-induced immunosuppression. The association between mHLA-DR and bacterial etiology may be linked to the clinical trajectory via differences in ability to cause intractable infection. Staphylococcus aureus was the dominating etiology among cases with unfavourable outcome. With focus on patients with S. aureus BSI, those with complicated S. aureus BSI were found to have lower HLA-DR mRNA expression during the first week than those with uncomplicated S. aureus BSI. If these results can be confirmed in a larger cohort, HLA-DR measurement could possibly become an additional tool for early identification of patients who require further investigation to clear infectious foci and achieve source control. In conclusion, PCR-based measurement of HLA-DR is a promising method for measurements of the immune state in BSI, but needs further evaluation in the intensive care unit setting to define the predictive and prognostic value for deleterious immunosuppression. The etiology of infection should be taken into consideration in future studies of translational immunology in sepsis.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

This study describes the development and validation of mutagenized cloned DNA constructs, which correspond to the polymorphic regions of the class II region of the HLA complex. The constructs were used to verify the allelic specificity of primers and probes in polymerase chain reaction (PCR)-based HLA typing assays such as Sequence Specific Primers (SSP) and Sequence Specific Oligonucleotide Probes (SSOP). The constructs consisted of the entire polymorphic region of exon 2 of class II HLA allele sequences that included primer annealing sites or probe hybridization sites. An HLA allele sequence was inserted into a plasmid, cloned, then mutagenized to match a specific HLA allele, and finally, the correct clone was verified by bidirectional sequencing of the insert. Thus, the construct created a cloned reference DNA sample for any specific allele, and can be used to validate the accuracy of various molecular methodologies.

La Sclérodermie Systémique (ScS) est une maladie auto-immune rare et complexe, cliniquement divisée en deux sous-groupes : la ScS cutanée diffuse (dc-ScS) et la ScS cutanée limitée (lc-ScS). Les anticorps anti-topoisomérase et anti-centromère (ATA et ACA) sont respectivement les marqueurs de chaque sous-groupe. Beaucoup d'études ont analysé les gènes des Antigènes leucocytaires humains (HLA), fort facteur de risque, dans plusieurs groupes ethniques de patients atteints de ScS. La plupart des allèles de susceptibilité HLA-DRB1 (*11:01, *11:04, *15:01, *08:02…) et DQB1 (*03:01, *03:02, *06:01 and *06:02) ont en commun une séquence d'acides aminés, respectivement 67FLEDR71 et 71TRAELDT77 sur leurs chaînes β. Pour la première fois, nous évaluons le risque relatif conféré par une ou deux doses de 67FLEDR71 et/ou une ou deux doses de 71TRAELDT77 chez des patients Caucasiens Français atteints de SSc. Nous montrons que le motif 67FLEDR71 est fortement associé à la dc-ScS et encore plus à la production d'ATA, alors que l'association de 71TRAELDT77 est plus faible dans les deux sous-groupes. Notre groupe a montré récemment dans la polyarthrite rhumatoïde (PR) que les patients qui n'ont pas les allèles de susceptibilité à la PR peuvent acquérir ces allèles via les cellules fœtales et/ou maternelles, aussi appelé Microchimérisme (Mc). Nous avons testé la même hypothèse dans la ScS. Contrairement à la PR, nos résultats n'ont pas démontré un tel transfert de susceptibilité via le Mc dans la SSc. Finalement, la présence d'auto-anticorps, liée à un génotype HLA, est précieuse au diagnostic et /ou pronostic de la maladie (ex : ACA ou ATA). / Systemic sclerosis (SSc) is a rare and complex autoimmune disease clinically divided into two subgroups: diffuse cutaneous (dcSSc) and limited cutaneous (lcSSc). Anti-topoisomerase and anti-centromere antibodies (ATA and ACA) are respectively markers of each subset. Many studies have analyzed Human Leukocyte Antigen (HLA) genes, the strongest genetic risk factor, in several ethnic groups in patients with SSc. Most common HLA-DRB1 (*11:01, *11:04, *15:01, *08:02…) and HLA-DQB1 (*03:01, *03:02, *06:01 and *06:02) susceptibility alleles have in common an amino acid sequence, respectively 67FLEDR71 and 71TRAELDT77 on their β chains. For the first time, we evaluate the relative risk conferred by one or two 67FLEDR71 and/or one or two 71TRAELDT77 motives among Caucasian French patients with SSc. We show that 67FLEDR71 motif is highly associated with dcSSc and even more with ATA production, whereas 71TRAELDT77 association is weaker in both subgroups. Our group has recently shown in Rheumatoid Arthritis (RA) that patients who lack the RA HLA susceptibility that it could be transferred to patients via fetal and/or maternal cells, also called microchimerism (Mc). We test the same hypothesis in SSc. Contrary to RA, our results fail to demonstrate such transfer of HLA susceptibility via Mc in SSc. Finally linked to a particular HLA genotype is the presence of autoantibodies, helpful for disease diagnosis and/or prognosis (i.e. ACA or ATA). Patients who do not have these markers are difficult to diagnose or classify. By ProtoArray® technique, we identify 6 new auto-antigens in the plasmas of SSc patients.

Humana leukocytantigen (HLA) är en typ av proteiner som uttrycks på alla cellers yta, de karakteriseras som antigenpresenterande och är en mekanism som ryggradsdjur utvecklat för att lokalisera infekterade eller defekta celler. HLA presenterar peptider från cellens insida för immunförsvaret, vilket i sin tur aktiverar andra delar av immunförsvaret eller inducerar apoptos om främmande peptider presenteras. HIV är ett virus som infekterar celler som uttrycker CD4 på ytan, så som makrofager och T-hjälparceller som ingår i immunförsvaret. Dessa sjunker i antal och immunförsvaret blir försvagat. Obehandlad HIV leder till AIDS, därför är bromsmediciner som Abacavir viktigt. Abacavir har visat goda resultat, dock drabbas 5-8% av hypersensitivitetsreaktioner. Forskare har visat att dessa reaktioner är starkt relaterade till HLA-B*57:01 allelen. Genom screening av denna allel kan behandling med Abacavir undvikas för denna grupp patienter. Syftet med denna studie var att jämföra två olika kit, Olerup SSP® HLA-B*57:01 och Inno-train HLA-READY GENE B57, för typning av HLA-B*57 i 20 olika DNA prover. Dessa jämfördes med den befintliga metoden för HLA-typning som används på klinisk immunologi och transfusionsmedicin, Universitetssjukhuset, Lund. Jämförelsen grundade sig på skillnader mellan kiten, både vad gäller resultat, kostnader och analystider.  Proverna analyserades med PCR innan de separerades på agarosgel med gelelektrofores. Resultaten tolkades enligt tillverkarnas anvisningar. Resultaten visade att båda metoderna kan typa HLA korrekt. Alla resultat med Olerup stämde. Däremot blev 8 av 20 prover fel vid första körningen med Inno-train men resultaten stämde efter omkörning. Avseende kostnader är Inno-train billigare per prov men Olerup skulle bli billigare långsiktigt. För att verkligen fastställa vilket kit som är mest lämpligt behövs fler analyser utföras. / Human leokocyte antigen (HLA), a protein present on the surface of our cells, is characterized as antigen-presenting a mechanism vertebrates have developed to locate infected or defect cells. HLA presents peptides which are brought from the cell's inside to be presented for the immune system. HIV is a virus that infects cells that express CD4 on the surface, such as macrophages and T-helper cells. When these are decreasing, the immune system gets weakened. Untreated HIV leads to AIDS, therefore are inhibiting pharmaceuticals like Abacavir important. Abacavir has shown good results, unfortunately 5-8% gets hypersensitivity-reactions. Scientists have shown that these reactions are strongly related to the HLA-B*57:01 allele. By screening for this allele, treatment with Abacavir could be avoided for this group of patients. The purpose of this study was to compare two different kits, Olerup SSP® HLA-B*57:01 and Inno-train HLA-READY GENE B57, for screening of HLA-B*57 in 20 different DNA samples. These have previously been typed with the established method at the clinical immunology and transfusion medicine, Lund University Hospital. The comparison was based on differences between the kits, both in terms of results, costs and analytical times. PCR was run before the samples were separated on gel with electrophoresis. The results were interpreted in accordance to the manufactures instructions. The results showed that both methods could type HLA correctly. All results from Olerup was correct. However, 8 out of 20 samples showed wrong results at the first run with Inno-train. These were correct after a second run. In terms of costs, Inno-train is cheaper per test, but Olerup would be cheaper in the long term. To really determine which kit is most suitable, more analyzes are required.

INTRODUÇÃO: O Pênfigo Vulgar é uma doença bolhosa crônica que acomente pele e mucosas. A perda de adesão epitelial ocorre por agressão autoimune às desmogleínas presentes nos desmossomos, mediada por anticorpos IgG. Estudos sobre a gênese da autoimunidade no pênfigo indicam associação entre alelos do sistema HLA, especialmente dos loci DR e DQ. A população brasileira apresenta características favoráveis a estudos exploratórios em genética decorrente de sua origem mista e intensa miscigenação. PACIENTES E MÉTODO: O grupo em estudo incluiu trinta e seis pacientes não consanguíneos com diagnóstico de Pênfigo Vulgar comprovado por imunopatologia provenientes do estado de São Paulo, Brasil. Foram tipados para os loci HLA-A, HLA-B e HLA-DR utilizando-se oligonucleotídeos sequência-específica (PCR-SSO). As frequências alélicas e fenotípicas encontradas foram comparadas com as de um grupo controle composto de dados de 712 indivíduos doadores voluntários cadastrados no Registro Nacional de Doadores de Medula Óssea (REDOME) provenientes de São Paulo e tipados pelo mesmo método. O valor de P crítico foi corrigido utilizando-se o método False Discovery Rate. RESULTADOS: Os alelos HLA-DRB1*04:02, DRB1*08:04 e DRB1*14 estiveram associados à doença com riscos relativos de 44,6, 18,6 e 4,8, respectivamente (p<0,001). Não houve diferença estatisticamente significante entre as frequências de nenhum alelo dos loci HLA-A ou HLA-B entre os grupos. DISCUSSÃO: O alelo DRB1*04:02, diretamente, e o alelo DRB1*14, indiretamente por desequilíbrio de ligação com DQB1*05:03, estão associados com Pênfigo Vulgar em diversas populações ao redor do mundo, porém nenhum estudo semelhante observou associação com o alelo DRB1*08:04 em tamanha magnitude. Acreditamos que as associações encontradas em nosso estudo não sejam decorrentes de viés de estratificação populacional. É necessária, no entanto, a tipagem de loci adjascentes ao HLA-DR dos indivíduos do grupo em estudo para diferenciar se o risco à doença é inerente a estes alelos ou a algum outro nas proximidades, com o qual estariam em desequilíbrio de ligação. CONCLUSÕES: Os alelos HLA-DRB1*04:02, DRB1*08:04 e DRB1*14 estiveram associados ao Pênfigo Vulgar em pacientes brasileiros. / BACKGROUND: Pemphigus vulgaris is a chronic blistering disease affecting skin and mucous membranes. Autoimmune aggression to desmoglein in desmosomes, mediated by IgG antibodies, leads to loss of epithelial cell adhesion. Studies indicate association between some alleles of the HLA system and pemphigus vulgaris, mainly at the DR and DQ loci. Brazilian population characteristics are conducive to genetic exploratory studies because of its various origins and intense ethnically admixture. PATIENTS AND METHODS: The study group consisted of thirty-six unrelated patients with clinical and immunopathological diagnosis of pemphigus vulgaris from a tertiary hospital in Sao Paulo - Brazil. HLA allele typing at the A, B and DR loci was performed after DNA extraction using polymerase chain reaction and sequence-specific oligonucleotide probes (PCR-SSO). Allele and phenotypic frequencies were compared to those from a control group composed by 712 individuals volunteer donors registered in a national registry of bone marrow donors (REDOME) from Sao Paulo, typed using the same method. False Discovery Rate method was used to adjust level of critical P values. RESULTS: The HLADRB1* 04:02, DRB1*08:04 and DRB1*14 were associated with pemphigus vulgaris with relative risks of 44.6, 18.6 and 4.8, respectively (p <0.001). There was no significant difference between the frequencies of any allele of loci HLAA or HLA-B among the groups. DISCUSSION: The alleles DRB1*04:02 and DRB1*14 (indirectly through linkage disequilibrium with the DQB1*05:03) are associated with pemphigus vulgaris in several populations worldwide, however, no similar study reported such magnitude of association between pemphigus vulgaris and DRB1*08:04 allele. We consider that the association is not secondary to population stratification bias. HLA typing of nearby loci is required to differentiate if the association with pemphigus vulgaris is inherent to the HLA-DRB1*08:04 allele or to another gene which is in linkage disequilibrium. CONCLUSIONS: The HLA-DRB1*04:02, DRB1*08:04 and DRB1*14 were associated with pemphigus vulgaris in Brazilian patients

A gravidez humana é permeada por diversos mecanismos de adaptação para evitar a rejeição do feto, que, por se tratar de um tecido semialogênico, deveria ser rejeitado pelo sistema imunológico materno. A molécula HLA-G, presente na interface materno-fetal, apresenta uma característica imunossupressora que age proporcionando inibição contra a citotoxidade das células NK e linfócitos T citotóxicos, conferindo efeito modulador benéfico à gestação. Entretanto, devido a suas propriedades imunossupressoras, essa molécula pode estar envolvida na persistência e progressão da infecção pelo HIV-1. Nosso objetivo foi avaliar a expressão das moléculas HLA-G em tecidos placentários de mulheres infectadas ou não pelo HIV-1. Trata-se de um estudo transversal e descritivo, para o qual foram coletadas biópsias placentárias de 100 mulheres infectadas pelo HIV-1 e 100 mulheres não infectadas. Ao realizar a análise dos dados sócio-demográficos, pudemos observar que o perfil das mulheres atendidas num hospital de referência no município de Ribeirão Preto assemelha-se ao encontrado nacionalmente. A faixa etária prevalente foi de 20 a 29 anos (48%); em relação à cor da pele, a grande maioria (71%) era branca; quanto à escolaridade, a população estudada apresentou maior frequência de ensino fundamental (61%); e a maioria (68%) não desempenhava atividade remunerada. Quanto à via de parto, 56,7% das mulheres foram submetidas a cesárea eletiva e 100% fizeram uso de antirretroviral injetável no momento do parto, conforme preconização do Ministério da Saúde. A técnica laboratorial utilizada foi imunoistoquímica, cuja análise foi realizada por um patologista. Os resultados obtidos não mostraram associação entre a expressão das moléculas HLA-G e os biomarcadores da infecção pelo HIV-1, como carga viral (cópias/mL) e células CD4+ (células/mm³), nem em relação ao uso de terapia antirretroviral (TARV). Contudo, observamos que as placentas das mulheres infectadas pelo HIV-1 apresentaram redução na expressão de HLA-G em comparação com as mulheres que não apresentam a infecção (p<0,01), sugerindo que essa redução possa ser um mecanismo de escape viral, que acarreta a redução da apresentação de peptídeos virais para os linfócitos T CD8+. Para melhor compreensão do padrão de expressão das moléculas HLA-G em placentas de mulheres infectadas pelo HIV-1, faz-se necessária a realização de outros estudos utilizando metodologias distintas / In order to prevent the rejection of the semiallogenic tissue of the fetus by the maternal immune system, human pregnancy presents a variety of adaptation mechanisms. The HLA-G molecule in the maternal-fetal interface, presents an immunosuppressant feature that inhibits the cytotoxicity of NK cells and T lymphocytes, with a positive modulating effect over the pregnancy. Nevertheless, due to its immunosuppressive properties, the HLA-G molecule might be related to the continuance and the progression of the HIV- 1 infection. Our objective was to analyse the expression of HLA-G in placental tissues of both HIV-1 infected and uninfected women. As a descriptive and cross-sectional study, placental biopsies were collected from 100 HIV-1 infected women and 100 from uninfected ones. When socio- demographic data were analysed, it was observed that the profile of women taken care at the referential hospital in Ribeirão Preto resembled the ones found nationwide: the most prevalent age group was the 20-29 years (48%); the vast majority was of white people (71%); there was a higher frequency of primary education in this group (61%); and most of them did not perform any paid activity (68%); 56.7% of them underwent elective cesarean; and 100 % of them made use of injectable antiretroviral at the delivery, as prescribed by the Ministry of Health. The laboratorial technique used was immunohistochemical, which was performed by a pathologist. Final results showed no association between the expression of HLA-G and the biomarkers of the HIV-1, such as viral load (copies/mL) and CD4+ cell counting (cells/mm³) or even the use of antiretroviral therapy (ART). Nevertheless, in the placenta of women infected with HIV-1, the expression of the HLA-G proved to be reduced in comparison to women who did not have the infection (p <0.01 ), suggesting that this reduction could be a mechanism for viral escape which entails the reduction of viral peptides to CD8+ T lymphocytes. To understand the full extension of the expression pattern of the HLA-G in HIV-1 infected women, it is necessary to perform further studies using different methodologies